Elevated plasma levels of homocysteine (Hcy) known as hyperhomocysteinemia (HHcy) cause vascular disease. The long-term objective of this project is to understand the mechanism of vascular remodeling in HHcy. Studies from the previous funding period showed that there is an inverse relationship between plasma Hcy and levels of peroxisome proliferators activated receptor (PPAR), a nuclear receptor which ameliorates vascular endothelial dysfunction. The central hypothesis of this competitive renewal application is that Hcy decreases thioredoxin, peroxiredoxin, increases NADH oxidase, mtNOS activity, and reactive oxygen species (ROS) in mitochondria in a gene dose-dependent manner. ROS transduces metalloproteinase activation causing thickening (fibrosis) of the basement membrane, rendering ineffective endothelial nitric oxide synthase (eNOS) and promotes endothelial-smooth muscle disconnection/uncoupling by antagonizing PPARy activity. The specific aims to address the central hypothesis are as following: Specific Aim #1: To determine whether by rendering ineffective PPARy Hcy decreases mitochondrial thioredoxin, peroxiredoxin, increases NADH oxidase, mtNOS activity and ROS. The PPARy agonist, ciglitazone will be administered to wild type (WT), cystathione (3 synthase (CBS) knockout heterozygote and homozygote (i.e. CBS +/+, CBS - /+ and CBS -/-), PPARy-/+, iNOS-/-, p47-/-, MMP-9-/- and double knockout mice. Aortic PPARy activity will be measured by EMSA. Levels of mitochondrial ROS will be measured using 2, 7-dichlorofluorescein in continuous assay. Peroxiredoxin, thioredoxin and NADH oxidase will be measured in situ, by Western blot and RT-PCR analyses. Specific Aim #2: To determine whether Hcy increases metalloproteinase activity, decreases the elastin/collagen ratio, and causes fibrosis by antagonizing PPARy. Metalloproteinase, TIMP, elastin and collagen will be measured in situ, by novel 2-D zymography, reverse-zymography, real-time RT- PCR and Western blot analysis. Specific Aim #3: To determine whether Hcy attenuates vascular function by increasing iNOS and rendering eNOS ineffective by antagonizing PPARy. Aortic contractile response will be measured. The novelty of this proposal is that it elucidates the mitochondrial mechanism of oxidative stress and vascular remodeling by Hcy. This study will provide new insights into the mechanism of arterial remodeling and will have therapeutic ramifications for vessel wall disease.